Extended Data Fig. 5: PKM2 activity increases glutamine derived OAA. Related to Fig. 5.
a. Isotopomer analysis of 13C6 glucose-derived carbon incorporation to OAA in RPMI 8226 cells treated with 13C6 glucose and 40 μM DASA or vehicle overnight. b. Isotopomer analysis of 13C5 glutamine-derived carbon incorporation to OAA in RPMI 8226 cells treated with 13C5 glutamine and 40 μM DASA or vehicle overnight. c. Western blot analysis with antibodies against GPT2 and β-actin of H1299 lysates from cells treated with vehicle or 40 μM DASA overnight. d. Relative OAA concentrations in H1299 GPT2 knockdown cells treated with vehicle or 40 μM DASA overnight. e. Western blot analysis of H1299 vector and PHGDH knockdown cell lysates with antibodies against PHGDH and β-actin. f. 13C lactate labeling rate of H1299 vector and PHGDH knockdown cells incubated with 13C6 glucose and vehicle or 40 μM DASA. g. 13C pyruvate labeling rate of H1299 vector and PHGDH knockdown cells incubated with 13C6 glucose and vehicle or 40 μM DASA. h. In situ LDH activity in H1299 vector and PHGDH knockdown cells treated with vehicle or 40 μM DASA. i. Western blot analysis of H1299 vector and PC knockdown cell lysates with antibodies against PC and β-actin. j. 13C lactate labeling rate of H1299 vector and PC knockdown cells incubated with 13C6 glucose and vehicle or 40 μM DASA. k. 13C pyruvate labeling rate of H1299 vector and PC knockdown cells incubated with 13C6 glucose and vehicle or 40 μM DASA. l. In situ LDH activity in H1299 vector and PC knockdown cells treated with vehicle or 40 μM DASA. Data is represented as the mean and error bars represent the standard deviation from n = 4 for (a), (d), (f), (g), (h), (j), (k), (l), n = 3 for (b) of biologically independent replicates. Western blot results in (c), (e), (i) are representative experiments of 3 biologically independent replicates. P values were determined by a two-tailed Student’s t-test.
